Activation of the control reporter plasmids pRL-TK and pRL-SV40 by multiple GATA transcription factors can lead to aberrant normalization of transfection efficiency

BACKGROUND: Members of the GATA transcription factor family have been used in many transfection studies to investigate their roles in the regulation of gene expression. To correct for variations in transfection efficiency, the Renilla luciferase encoding plasmids pRL-TK and pRL-SV40 are commonly used as normalization controls. RESULTS: We report here that plasmids expressing GATA-4 or GATA-6 transcription factor increased Renilla luciferase gene expression by 2 to 8 fold when co-transfected with pRL-TK or pRL-SV40. This alteration of the control reporter gene activity was shown to cause erroneous normalization of transfection efficiency and thus misinterpretation of results in a transactivation assay. To circumvent the problem, we generated two mutant control plasmids from which putative GATA response elements were deleted. These deletions rendered pRL-SV40 unresponsive to GATA transcription factor stimulation and reduced the response of pRL-TK. A database search also indicates that consensus GATA binding sequences are present in other commercially available Renilla luciferase encoding plasmids; therefore, the latter can potentially be transactivated by GATA transcription factors. CONCLUSION: Taken together, these findings highlight the importance of the selection of an appropriate control reporter plasmid for the normalization of transfection efficiency

Racial Differences in Genetic and Environmental Risk to Preterm Birth

Preterm birth is more prevalent in African Americans than European Americans and contributes to 3.4 times more African American infant deaths. Models of social inequity do not appreciably account for this marked disparity and molecular genetic studies have yet to characterize whether allelic differences that exist between races contribute to this gap. In this study, biometrical genetic models are applied to a large mixed-race sample consisting of 733,339 births to measure the extent that heritable factors and environmental exposures predict the timing of birth and explain differences between racial groups. Although we expected significant differences in mean gestational age between racial groups, we did not anticipate the variance of gestational age in African Americans (σ2 = 7.097) to be nearly twice that of European Americans (σ2 = 3.764). Our results show that this difference in the variance of gestational age can largely be attributed to environmental sources; which were 3.1 times greater in African Americans. Specifically, environmental factors that change between pregnancies, versus exposures that influence all pregnancies within a family, are largely responsible for the increased reproductive heterogeneity observed in African American mothers. Although the contribution of both fetal and maternal genetic factors differed between race categories, genetic studies may best be directed to understanding the differences in the socio-cultural sources of this heterogeneity, and their possible interaction with genetic differences within and between races. This study provides a comprehensive description of the relative genetic and environmental contributions to racial differences in gestational age

Modeling Longitudinal Change in Cervical Length Across Pregnancy

Introduction: A short cervix (cervical length \u3c 25 mm) in the mid-trimester (18 to 24 weeks) of pregnancy is a powerful predictor of spontaneous preterm delivery (gestational age at delivery \u3c 37 weeks). Although the biological mechanisms of cervical remodeling have been the subject of extensive investigation, very little is known about the rate of change in cervical length over the course of a pregnancy, or the extent to which rapid cervical shortening increases maternal risk for spontaneous preterm delivery. Methods: A cohort of 5,160 unique women carrying 5,971 singleton pregnancies provided two or more measurements of cervical length during pregnancy. Cervical length was measured in millimeters using a transvaginal 12-3 MHz ultrasound endocavity probe (SuperSonic Imagine). Maternal characteristics, including relevant medical history and birth outcome data, were collected for each participant. Gestational age at delivery was measured from the first day of each woman’s last menstrual period and confirmed by ultrasound. Repeated measurements of cervical length during pregnancy were modeled as a longitudinal, multilevel growth curve in MPlus. A three-level variance structure was used to account for non-independence of repeated measurements clustered within pregnancies, which are clustered within participants. Results: The average number of cervical length measurements per pregnancy is 6. Shorter mid-trimester cervical lengths and accelerated rates of cervical shortening are associated with shorter gestational duration. A smaller initial cervical length (p \u3c 1*10-4) and a faster rate of change in cervical change length during pregnancy (p \u3c 1*10-4) are significantly associated with an earlier gestational age at delivery. A higher pre-pregnancy body mass index (BMI) is associated with shorter initial cervical length in early pregnancy (p \u3c 1*10-4), while maternal age is associated with a more rapid rate of change in cervical length (p \u3c 1*10-4). Parameters describing cervical length and its rate change during pregnancy (i.e., intercept, linear slope, and quadratic slope parameters) explained 59% more variance in gestational age at delivery than a single mid-rimester cervical length measurement, which is the current gold standard in clinical practice. However, a significant amount of residual variance in individual estimates of cervical length growth parameters remains (p \u3c 1*10-4), which could be accounted for, in part, by common variation in the population. Conclusion: We have developed longitudinal models of cervical length that describe individual and group level trajectories of cervical change across pregnancy. Extensions of this model incorporating genomic data, can be used to estimate the heritability of cervical length and its role in mediating the timing of birth.https://scholarscompass.vcu.edu/gradposters/1141/thumbnail.jp

Rare mutations and potentially damaging missense variants in genes encoding fibrillar collagens and proteins involved in their production are candidates for risk for preterm premature rupture of membranes

Preterm premature rupture of membranes (PPROM) is the leading identifiable cause of preterm birth with ~ 40% of preterm births being associated with PPROM and occurs in 1% - 2% of all pregnancies. We hypothesized that multiple rare variants in fetal genes involved in extracellular matrix synthesis would associate with PPROM, based on the assumption that impaired elaboration of matrix proteins would reduce fetal membrane tensile strength, predisposing to unscheduled rupture. We performed whole exome sequencing (WES) on neonatal DNA derived from pregnancies complicated by PPROM (49 cases) and healthy term deliveries (20 controls) to identify candidate mutations/variants. Genotyping for selected variants from the WES study was carried out on an additional 188 PPROM cases and 175 controls. All mothers were self-reported African Americans, and a panel of ancestry informative markers was used to control for genetic ancestry in all genetic association tests. In support of the primary hypothesis, a statistically significant genetic burden (all samples combined, SKAT-O p-value = 0.0225) of damaging/potentially damaging rare variants was identified in the genes of interest—fibrillar collagen genes, which contribute to fetal membrane strength and integrity. These findings suggest that the fetal contribution to PPROM is polygenic, and driven by an increased burden of rare variants that may also contribute to the disparities in rates of preterm birth among African Americans

In memoriam: Celso-Ramon Garcia, M.D. (1922–2004), reproductive medicine visionary

This article traces the career of Celso-Ramon Garcia (1922–2004), noted physician, educator, and internationally renowned pioneer in the field of reproductive endocrinology. His work helped to formulate oral contraceptives used by millions of women throughout the world. Garcia's research collaborators included Gregory Pincus and John Rock, who together finalized the landmark clinical data needed to secure initial FDA approval for "the pill" in 1960. In addition to Garcia's monumental work in contraceptive endocrinology, his scholarly interests encompassed physiology of the menopause, minimally invasive reproductive surgery, as well as psychological aspects of infertility. Closely identified with the University of Pennsylvania, Garcia was instrumental in establishing the first formal clinical program in reproductive biology and influenced countless young scientists whose training he supervised and mentored. His distinguished career was emblematic of the best of the medical profession, characterized by compassion, intellect, and a sincere desire to help others. Our manuscript outlines Garcia's wide range of interests, acknowledges his superior fund of knowledge, and honors his humanitarian spirit – all of which contributed to an impressive legacy of medical discoveries. The impact of Prof. Garcia's work will continue to be felt for many years

Prospective Longitudinal Study of the Pregnancy DNA Methylome: The US Pregnancy, Race, Environment, Genes (PREG) Study

Purpose The goal of the Pregnancy, Race, Environment, Genes study was to understand how social and environmental determinants of health (SEDH), pregnancy-specific environments (PSE) and biological processes influence the timing of birth and account for the racial disparity in preterm birth. The study followed a racially diverse longitudinal cohort throughout pregnancy and included repeated measures of PSE and DNA methylation (DNAm) over the course of gestation and up to 1 year into the postpartum period. Participants All women were between 18 and 40 years of age with singleton pregnancies and no diagnosis of diabetes or indication of assisted reproductive technology. Both mother and father had to self-identify as either African-American (AA) or European-American (EA). Maternal peripheral blood samples along with self-report questionnaires measuring SEDH and PSE factors were collected at four pregnancy visits, and umbilical cord blood was obtained at birth. A subset of participants returned for two additional postpartum visits, during which additional questionnaires and maternal blood samples were collected. The pregnancy and postpartum extension included n=240 (AA=126; EA=114) and n=104 (AA=50; EA=54), respectively. Findings to date One hundred seventy-seven women (AA=89, EA=88) met full inclusion criteria out of a total of 240 who were initially enrolled. Of the 63 participants who met exclusion criteria after enrolment, 44 (69.8%) were associated with a medical reason. Mean gestational age at birth was significantly shorter for the AA participants by 5.1 days (M=272.5 (SD=10.5) days vs M=277.6 (SD=8.3)). Future plans Future studies will focus on identifying key environmental factors that influence DNAm change across pregnancy and account for racial differences in preterm birth

Otitis Media in Sperm-Associated Antigen 6 (Spag6)-Deficient Mice

Mammalian SPAG6 protein is localized to the axoneme central apparatus, and it is required for normal flagella and cilia motility. Recent studies demonstrated that the protein also regulates ciliogenesis and cilia polarity in the epithelial cells of brain ventricles and trachea. Motile cilia are also present in the epithelial cells of the middle ear and Eustachian tubes, where the ciliary system participates in the movement of serous fluid and mucus in the middle ear. Cilia defects are associated with otitis media (OM), presumably due to an inability to efficiently transport fluid, mucus and particles including microorganisms. We investigated the potential role of SPAG6 in the middle ear and Eustachian tubes by studying mice with a targeted mutation in theSpag6 gene. SPAG6 is expressed in the ciliated cells of middle ear epithelial cells. The orientation of the ciliary basal feet was random in the middle ear epithelial cells of Spag6-deficient mice, and there was an associated disrupted localization of the planar cell polarity (PCP) protein, FZD6. These features are associated with disordered cilia orientation, confirmed by scanning electron microscopy, which leads to uncoordinated cilia beating. The Spag6 mutant mice were also prone to develop OM. However, there were no significant differences in bacterial populations, epithelial goblet cell density, mucin expression and Eustachian tube angle between the mutant and wild-type mice, suggesting that OM was due to accumulation of fluid and mucus secondary to the ciliary dysfunction. Our studies demonstrate a role for Spag6 in the pathogenesis of OM in mice, possibly through its role in the regulation of cilia/basal body polarity through the PCP-dependent mechanisms in the middle ear and Eustachian tubes

Estrogen Metabolites in Human Corpus Luteum Physiology: Differential Effects on Angiogenic Activity

OBJECTIVE: To determine tissue concentrations of E2, estrone, P, and estrogens metabolites (EMs) 2-methoxyestradiol, 2-methoxyestrone, 4-hydroxyestrone, and 16-ketoestradiol in corpus luteum (CL) of different ages, and after hCG administration; and to examine the effects of EMs on vascular endothelial growth factor (VEGF) secretion and angiogenic activity released by cultured luteinizing granulosa cells in the presence and absence of hCG. DESIGN: Experimental study. SETTING: University. PATIENT(S): Thirty-two healthy women of reproductive age. INTERVENTION(S): Corpus luteum was collected at the time of minilaparotomy for tubal sterilization, at varying stages of the luteal phase (LP). Late-LP CL was collected 24 hours after IM administration of 10,000 IU hCG. Granulosa cells were isolated from follicular aspirates obtained from healthy women participating in our IVF program for male factor infertility. MAIN OUTCOMES MEASURE(S): Estrogen metabolite concentrations were determined in CL tissue, and VEGF was assessed in conditioned medium. The angiogenic activity was analyzed by bioassay. RESULT(S): Concentrations of EMs with proangiogenic activity (16-ketoestradiol and 4-hydroxyestrone) were higher in early and mid-LP CL vs. late-LP CL. These EMs and hCG increased VEGF production and angiogenic activity. Conversely, late-LP CL had significantly higher levels of 2-methoxyestrone and 2-methoxyestradiol, which have antiangiogenic activity. Administration of hCG reduced the production of these EMs. CONCLUSION(S): Our findings suggest that the EMs are important paracrine modulators of CL function. Administration of hCG increases the production of EMs with proangiogenic activity and reduces the secretion of those EMs with antiangiogenic action, suggesting a novel mechanism by which the late-LP CL is rescued in conception cycles

Reassessing the impact of smoking on preeclampsia/eclampsia: Are there age and racial differences?

To investigate the association between cigarette use during pregnancy and pregnancy-induced hypertension/preeclampsia/eclampsia (PIH) by maternal race/ethnicity and age.This retrospective cohort study was based on the U.S. 2010 natality data. Our study sample included U.S. women who delivered singleton pregnancies between 20 and 44 weeks of gestation without major fetal anomalies in 2010 (n = 3,113,164). Multivariate logistic regression models were fit to estimate crude and adjusted odds ratios and the corresponding 95% confidence intervals.We observed that the association between maternal smoking and PIH varied by maternal race/ethnicity and age. Compared with non-smokers, reduced odds of PIH among pregnant smokers was only evident for non-Hispanic white and non-Hispanic American Indian women aged less than 35 years. Non-Hispanic Asian/Pacific Islander women who smoked during pregnancy had increased odds of PIH regardless of maternal age. Non-Hispanic white and non-Hispanic black women 35 years or older who smoked during pregnancy also had increased odds of PIH.Our study findings suggest important differences by maternal race/ethnicity and age in the association between cigarette use during pregnancy and PIH. More research is needed to establish the biologic and social mechanisms that might explain the variations with maternal age and race/ethnicity that were observed in our study